Method for producing proteins in transformed Pichia

Abstract

Methylotrophic yeasts are useful hosts for the production of commercially valuable recombinant proteins. However, the development of large-scale cultures of recombinant methylotrophic yeasts has been hindered by the formation of precipitation in culture media. A new soluble minimal medium overcomes this problem. Moreover, new feeding schemes provide cultures of high biomass, which produce biologically active recombinant protein.

Description

The present invention relates generally to methods for producing heterologous proteins in transformed cells. In particular, the present invention provides improved methods for culturing transformed methylotrophic yeast cells that express heterologous proteins.Certain yeasts are able to utilize methanol as a sole source of carbon and energy. Species of the so-called methylotrophic yeasts that have the biochemical pathways necessary for methanol utilization are classified into four genera, based upon cell morphology and growth characteristics: Hansenula, Pichia, Candida, and Torulopsis (Billon-Grand, Mycotaxon 35:201 (1989); Kurtzman, Mycologia 84:72 (1992)). Not all species within these genera are capable of utilizing methanol as a source of carbon and energy, and therefore, individual species of a genus may differ in physiology and metabolism.Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems. Some methylotrophic yeasts have been shown ...

AI Technical Summary

Benefits of technology

The present invention provides improved methods for producing a peptide or polypeptide by a recombinant methylotrophic yeast host.

An "anti-idiotype antibody" is an antibody that binds with the variable region domain of an immunoglobulin. As a result, an anti-idiotype antibody can mimic the epitope that binds with the variable region of the immunoglobulin.

Strains of Pichia methanolica are available from the American Type Culture Collection (Manassas, Va., USA) and other repositories. Cells to be transformed with heterologous DNA may have a mutation that can be complemented by a gene (a "selectable marker") on the heterologous DNA molecule. This selectable marker allows the transformed cells to grow under conditions in which untransformed cells cannot multiply ("selective conditions"). The general principles of selection are well known in the art. Commonly used selectable markers are genes that encode enzymes required for the synthesis of arnino acids or nucleotides. Auxotrophic mutants having mutations in these genes cannot grow in media lacking the specific amino acid or nucleotide unless the mutation is complemented by the selectable marker. Use of such selective culture media ensures the stable maintenance of the heterologous DNA within the host cell.

The DNA constructs used within the present invention may further contain additional elements, such as an origin of replication and a selectable marker that allow amplification and maintenance of the DNA in an alternate host (e.g., E. coli). To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment, comprising the promoter-gene of interest-terminator plus selectable marker, flanked at both ends by host DNA sequences. This is conveniently accomplished by including 3' untranslated DNA sequence at the downstream end of the expression segment and relying on the promoter sequence at the 5' end. When using linear DNA, the expression segment will be flanked by cleavage sites to allow for linearization of the molecule and separation of the expression segment from other sequences (e.g., a bacterial origin of replication and selectable marker). Preferred cleavage sites are those that are recognized by restriction endonucleases that cut infrequently within a DNA sequence, such as those that recognize eight-base target sequences (e.g., NotI).

Differences in expression levels of heterologous proteins can result from such factors as the site of integration and copy number of the expression cassette and differences in promoter activity among individual isolates. It is therefore advantageous to screen a number of isolates for expression level prior to selecting a production strain. A variety of suitable screening methods are available. For example, transformant colonies are grown on plates that are overlayed with membranes (e.g., nitrocellulose) that bind protein. Proteins are released from the cells by secretion or following lysis, and bind to the membrane. Bound protein can then be assayed using known methods, including immunoassays. More accurate analysis of expression levels can be obtained by culturing cells in liquid media and analyzing conditioned media or cell lysates, as appropriate. Methods for concentrating and purifying proteins from media and lysates will be determined in part by the protein of interest. Such methods are readily selected and practiced by the skilled practitioner.

Problems solved by technology

Development of methylotrophic yeasts as hosts for use in recombinant production systems has been slow, due in part to a lack of efficient promoters, selectable markers, and mutant host cells, as well as suitable transformation techniques.

The use of the expensive carbon source glycerol for Pichia methanolica is not practical due to the yeast's poor growth on this substrate.

One of the main drawbacks in using phosphate glass is that this component must be added separately as a filter-sterilized solution to the fermentor, and the basal medium may form a precipitate before addition of the phosphate glass.

The other major drawback is that phosphate glass can inhibit the growth of P. methanolica.

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